TP53BP1 Knockout MDA-MB-231 Cell Line
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Product ID： LM02025067369
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隐藏域元素占位
- 产品描述
- 细胞复苏
- 细胞传代
- 细胞冻存
- 抗体验证结果
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- Commodity name: TP53BP1 Knockout MDA-MB-231 Cell Line
- Commodity ID: LM02025067369
- Gene Symbol: TP53BP1
- Ensembl ID: ENSG00000067369
- Uniprot ID: Q12888
- 宿主细胞 / 类型: MDA-MB-231/human breast cancer cells
- NCBI Gene ID: 7158
- 规格: 1×10^6 cells/frozen vial
- 生长培养基: L-15+10%FBS+1%P,S
- 筛选标记: N/A
- 生长特性: Adherent cells, epithelial-like
- 培养条件: 37℃, air incubation, passaging at 1/2 to 1/4 confluency
- 倍增时间: ~40-60 hours
- 参考换液频率: 2–3 times/week
- 支原体检测结果: Negative
- 敲除效率（Sanger测序）: 100%
- 蛋白质组验证结果: Protein-level validation has been completed.
- 抗体货号: Adding...
- 目标基因介绍: Double-strand break (DSB) repair protein involved in response to DNA damage, telomere dynamics and class-switch recombination (CSR) during antibody genesis (PubMed:12364621, PubMed:22553214, PubMed:23333306, PubMed:17190600, PubMed:21144835, PubMed:28241136). Plays a key role in the repair of double-strand DNA breaks (DSBs) in response to DNA damage by promoting non-homologous end joining (NHEJ)-mediated repair of DSBs and specifically counteracting the function of the homologous recombination (HR) repair protein BRCA1 (PubMed:22553214, PubMed:23727112, PubMed:23333306). In response to DSBs, phosphorylation by ATM promotes interaction with RIF1 and dissociation from NUDT16L1/TIRR, leading to recruitment to DSBs sites (PubMed:28241136). Recruited to DSBs sites by recognizing and binding histone H2A monoubiquitinated at 'Lys-15' (H2AK15Ub) and histone H4 dimethylated at 'Lys-20' (H4K20me2), two histone marks that are present at DSBs sites (PubMed:23760478, PubMed:28241136, PubMed:17190600). Required for immunoglobulin class-switch recombination (CSR) during antibody genesis, a process that involves the generation of DNA DSBs (PubMed:23345425). Participates in the repair and the orientation of the broken DNA ends during CSR (By similarity). In contrast, it is not required for classic NHEJ and V(D)J recombination (By similarity). Promotes NHEJ of dysfunctional telomeres via interaction with PAXIP1 (PubMed:23727112).
- 细胞开发路径: A stable KO cell line was generated using the CRISPR-RNP method; Sanger sequencing confirmed 100% knockout efficiency in the KO cell line.
- 应用: Gene knockout cell lines (KO Cell Lines) with high knockout efficiency are particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad gene discovery studies.
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01. Pre-warm the complete culture medium in a 37°C water bath.
02. Thaw the cryovial in a 37°C water bath for 1–2 minutes.
03. Transfer the cryovial to a biosafety cabinet and wipe its surface with 70% ethanol.
04. Unscrew the cap of the cryovial and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete culture medium.
05. Centrifuge at 125g for 5–7 minutes at room temperature, then discard the supernatant.
06. Resuspend the cell pellet in 5 mL of complete culture medium and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to a 37°C, 5% CO2 incubator for culture.
08. Recommended passaging ratio: Passaging at a ratio of 1/2 to 1/4; cells will confluence in 2–3 days. -
01. Cell passage can be performed when the cell confluence in the culture flask reaches 80%–90% or higher.
02. Remove the culture medium, PBS, and trypsin (0.25% Trypsin-EDTA Gibco 25200-056) from the 4°C refrigerator, place them in a 37°C water bath, and remove them when their temperature approaches 37°C. After spraying the surfaces of the bottles with 75% ethanol, transfer them to a biosafety cabinet.03. Remove the culture flask to be passaged from the incubator, spray the exterior of the flask with 75% ethanol, and place it inside a biosafety cabinet.
04. To avoid detaching the cells, rinse them with PBS along the inner wall of the culture flask; discard the wash solution after rinsing, and add 2 mL to a T25 flask.
Add the appropriate volume of trypsin (1.5 mL for a T75 flask, 0.5 mL for a T25 flask), and gently swirl the flask so the trypsin evenly coats the bottom. Adjust the volume as needed. After approximately 1–2 minutes, when most cells have detached, add the corresponding volume of complete culture medium to stop the digestion, and gently pipette up and down with a 5 mL pipette until all cells are fully detached.
06. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300g for 5 min, and discard the supernatant.
07. Resuspend the cells in 5 mL of complete medium, adjust the seeding ratio as needed, and replenish the complete medium in the culture flasks—add up to 13–15 mL for T75 flasks and 5 mL for T25 flasks—and supplement with 1% double antibiotic.
08. After capping the bottle and tightening the cap, gently shake the bottle to mix the cells evenly, then place it in a 37°C, 5% CO2 incubator. -
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements; examine the following conditions under a microscope: healthy appearance and morphological characteristics, appropriate growth phase (late logarithmic phase), and absence of contamination or signs of decline.
03. Digest and centrifuge the cells (refer to the subculture protocol for specific steps).
04. Resuspend the cells in cryopreservation solution at 1 mL per tube, mix thoroughly by pipetting, and aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a -80°C freezer.
06. The cells are subsequently transferred to a liquid nitrogen tank for long-term storage. - Western blot (WB) analysis was performed simultaneously on lysates from wild-type and KO cells using a specific monoclonal antibody; the target protein band was detected in WT cells but absent or diminished in KO cells.
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Classification： Gene-knockout cell line (protein levels validated)
Cell Line Information
Gene Symbol
TP53BP1
NCBI Gene ID
7158
Ensembl ID
ENSG00000067369
Uniprot ID
Q12888
Screening marker
N/A
Host cell/type
MDA-MB-231/human breast cancer cells
Specifications
1×10^6 cells/frozen vial
Growth Medium
L-15+10%FBS+1%P,S
growth characteristics
Adherent cells, epithelial-like
culture condition
37℃, air incubation, passaging at 1/2 to 1/4 confluency
doubling time
~40-60 hours
Reference fluid change frequency
2–3 times/week
Mycoplasma test results
Negative
Knock-out validation
Knockout efficiency (Sanger sequencing)
100%
Proteome Validation Results
Protein-level validation has been completed.
Antibody number
Adding...
Antibody validation results
Cell Line Description
Introduction of target gene
Double-strand break (DSB) repair protein involved in response to DNA damage, telomere dynamics and class-switch recombination (CSR) during antibody genesis (PubMed:12364621, PubMed:22553214, PubMed:23333306, PubMed:17190600, PubMed:21144835, PubMed:28241136). Plays a key role in the repair of double-strand DNA breaks (DSBs) in response to DNA damage by promoting non-homologous end joining (NHEJ)-mediated repair of DSBs and specifically counteracting the function of the homologous recombination (HR) repair protein BRCA1 (PubMed:22553214, PubMed:23727112, PubMed:23333306). In response to DSBs, phosphorylation by ATM promotes interaction with RIF1 and dissociation from NUDT16L1/TIRR, leading to recruitment to DSBs sites (PubMed:28241136). Recruited to DSBs sites by recognizing and binding histone H2A monoubiquitinated at 'Lys-15' (H2AK15Ub) and histone H4 dimethylated at 'Lys-20' (H4K20me2), two histone marks that are present at DSBs sites (PubMed:23760478, PubMed:28241136, PubMed:17190600). Required for immunoglobulin class-switch recombination (CSR) during antibody genesis, a process that involves the generation of DNA DSBs (PubMed:23345425). Participates in the repair and the orientation of the broken DNA ends during CSR (By similarity). In contrast, it is not required for classic NHEJ and V(D)J recombination (By similarity). Promotes NHEJ of dysfunctional telomeres via interaction with PAXIP1 (PubMed:23727112).
Cell development path
A stable KO cell line was generated using the CRISPR-RNP method; Sanger sequencing confirmed 100% knockout efficiency in the KO cell line.
Application
Gene knockout cell lines (KO Cell Lines) with high knockout efficiency are particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad gene discovery studies.
Cell Culture Instructions
Cell Resuscitation
01. Pre-warm the complete culture medium in a 37°C water bath.
02. Thaw the cryovial in a 37°C water bath for 1–2 minutes.
03. Transfer the cryovial to a biosafety cabinet and wipe its surface with 70% ethanol.
04. Unscrew the cap of the cryovial and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete culture medium.
05. Centrifuge at 125g for 5–7 minutes at room temperature, then discard the supernatant.
06. Resuspend the cell pellet in 5 mL of complete culture medium and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to a 37°C, 5% CO2 incubator for culture.
08. Recommended passaging ratio: Passaging at a ratio of 1/2 to 1/4; cells will confluence in 2–3 days.
cell passage
01. Cell passage can be performed when the cell confluence in the culture flask reaches 80%–90% or higher.
02. Remove the culture medium, PBS, and trypsin (0.25% Trypsin-EDTA Gibco 25200-056) from the 4°C refrigerator, place them in a 37°C water bath, and remove them when their temperature approaches 37°C. After spraying the surfaces of the bottles with 75% ethanol, transfer them to a biosafety cabinet.
03. Remove the culture flask to be passaged from the incubator, spray the exterior of the flask with 75% ethanol, and place it inside a biosafety cabinet.
04. To avoid detaching the cells, rinse them with PBS along the inner wall of the culture flask; discard the wash solution after rinsing, and add 2 mL to a T25 flask.
Add the appropriate volume of trypsin (1.5 mL for a T75 flask, 0.5 mL for a T25 flask), and gently swirl the flask so the trypsin evenly coats the bottom. Adjust the volume as needed. After approximately 1–2 minutes, when most cells have detached, add the corresponding volume of complete culture medium to stop the digestion, and gently pipette up and down with a 5 mL pipette until all cells are fully detached.
06. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300g for 5 min, and discard the supernatant.
07. Resuspend the cells in 5 mL of complete medium, adjust the seeding ratio as needed, and replenish the complete medium in the culture flasks—add up to 13–15 mL for T75 flasks and 5 mL for T25 flasks—and supplement with 1% double antibiotic.
08. After capping the bottle and tightening the cap, gently shake the bottle to mix the cells evenly, then place it in a 37°C, 5% CO2 incubator.
cell cryopreservation
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements; examine the following conditions under a microscope: healthy appearance and morphological characteristics, appropriate growth phase (late logarithmic phase), and absence of contamination or signs of decline.
03. Digest and centrifuge the cells (refer to the subculture protocol for specific steps).
04. Resuspend the cells in cryopreservation solution at 1 mL per tube, mix thoroughly by pipetting, and aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a -80°C freezer.
06. The cells are subsequently transferred to a liquid nitrogen tank for long-term storage.
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